The overall objective of this research proposal is to examine in detail the electrophysiology of carrier-mediated H+ transport by the urinary bladder of the freshwater turtle, Pseudemys scripta using recently developed electrophysiologic techniques. The specific aims are to: (i) identify in vitro the microplicated epithelial cells responsible for active proton secretion by this epithelium; (ii) define the properties of the apical and baso-lateral membranes of these specialized cells under conditions of normal transport and following stimulation or inhibition of acid secretion; (iii) to define the electrical and chemical driving forces operating across the apical and baso-lateral membrane; (iv) to define the kinetic relations between the ionic flows across these barriers and their conjugate driving forces; and (v) to determine the mechanisms responsible for regulating the properties of the apical and baso-lateral membranes under conditions known to alter the rate of transcellular proton secretion. To accomplish these goals high resolution phase contrast and epifluorescent microscopy and micro-electrophysiologic techniques will be employed simultaneously to identify and impale individual H+-secreting cells. Techniques developed previously by the PI in collaboration with Dr. S. G. Schultz will be used to generate the current-voltage relations for H+ secretion across the apical lmembrane and HCO3- exit across the baso-lateral membrane. Cytoplasmic pH of these cells will be measured using both ion-selective microelectrodes and fluorescent probes. The results of these proposed studies will markedly advance our understanding of the mechanisms of H+ transport and its regulation at the membrane level.